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318 Thibodeau-Beganny and Joungbeen outlined in previous publications (12,13), the overall approach has evolvedin our laboratory as we have gained experience with the method. The protocoldescribed in detail herein is the most up-to-date method currently utilized bythe laboratory for selections.2. Materials2.1. Molecular Biology Reagents10X Annealing buffer (1 mL): 400 µL 1 M Tris-HCl, pH 8.0 200 µL 1 MMgCl 2, 100 µL 5 M NaCl, and 300 µL H 2O.2.2. Primer Sequences1. OK.181 sequencing primer: 5′-CCAGAGCATGTATCATATGGTCCAGAAACCC-3′.2. OK.5 polymerase chain reaction (PCR) primer: 5′-AAAATAGGCGTATCAC-GAGGCCCT-3′.3. OK.163 PCR primer: 5′-CGCCAGGGTTTTCCCAGTCACGAC-3′.4. OK.61 sequencing primer: 5′-GGGTAGTACGATGACGGAACCTGTC-3′.2.3. Bacterial Strains1. CSH100 (genotype: F′ lac proA + B + [lacI q lacPL8]/ara − ∆ [gpt-lac]5).2. KJ1C (genotype: F − ∆hisB463 ∆ [gpt-proAB-arg-lac] XIII zaj::Tn10).These strains are both available from the Joung laboratory (MassachusetteGeneral Hospital).2.4. Bacterial Media1. Luria Bertani (LB)/TKS plates contain tetracycline, kanamycin, and sucrose.2. LB/TK plates contain tetracycline and kanamycin.3. LB/Kan plates contain kanamycin.4. LB/CCK plates contain carbenicillin, chloramphenicol, and kanamycin.5. LB/CK plates contain chloramphenicol and kanamycin.6. LB/Tet plates contain tetracycline.7. M9 minimal medium plates.For 500 mL: autoclave 439 mL H 2O with 7.5 g bacto agar and magnetic stir bar.After agar has cooled to approx 65°C, add 50 mL 10X M9 salts, 1 mL 1 M MgSO 4,10 mL 20% glucose, and 0.5 mL 100 mM CaCl 2and then pour plates.8. NM medium (1 L): 836 mL H 2O, 100 mL 10X M9 salts, 20 mL 20% glucose, 10 mL20 mM adenine, 30 mL 200X amino acid mixture (see Step 10), 1 mL 1 M MgSO 4,1 mL 10 mg/mL thiamine, 1 mL 10 mM ZnSO 4, and 1 mL 100 mM CaCl 2.a. Filter-sterilize and store at 4°C.b. Add antibiotics, isopropyl-β-D-thiogalactopyranoside (IPTG), and 3-aminotriazole(3-AT) as desired.9. NM agar plates (1 L).a. Autoclave 836 mL H 2O, 15 g bacto agar, and a magnetic stir bar together.